?url_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Adc&rft.relation=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F19672%2F&rft.title=Multiequilibria+of+Oligomeric+Thermophilic+DNA+Replication+Polymerases&rft.creator=Lin%2C+Hsiang-Kai&rft.description=DNA+polymerases+are+essential+enzymes+in+all+domains+of+life+for+both+DNA+replication+and+repair.+We+examined+the+thermodynamics+and+enzymatic+activity+related+to+the+oligomerization+of+hyperthermophilic+archaeal+Sulfolobus+solfataricus+(Sso)+primary+DNA+replication+polymerase+(Dpo1)+and+lesion+bypass+polymerase+(Dpo4).+Both+Dpo1+and+Dpo4+bind+to+DNA+with+initial+high+affinity+monomeric+binding+followed+by+sequential+binding+of+additional+molecules+at+higher+concentrations+of+the+enzyme.+Gel+filtration%2C+chemical+crosslinking%2C+isothermal+titration+calorimetry+(ITC)+and+fluorescence+anisotropy+experiments+all+show+a+stoichiometry+of+three+Dpo1+and+two-four+Dpo4+molecules+bound+to+a+single+DNA+substrate.+In+particular%2C+oligomeric+Dpo1-DNA+complexes+significantly+increase+both+the+kinetic+rate+and+processivity+of+DNA+synthesis.+%0D%0ADifferentiation+of+binding+accurate+DNA+replication+polymerase+Dpo1+over+error+prone+DNA+lesion+bypass+polymerase+Dpo4+is+essential+for+the+proper+maintenance+of+the+genome.+Binding+discrimination+between+these+polymerases+on+DNA+templates+is+complicated+by+the+fact+that+multiple+oligomeric+species+are+influenced+by+concentration+and+temperature.+Fluorescence+anisotropy+experiments+were+used+to+separate+discrete+binding+events+for+the+formation+of+trimeric+Dpo1+and+dimeric+Dpo4+complexes+on+DNA.+The+associated+equilibria+are+found+to+be+temperature-dependent%2C+generally+leading+to+more+favorable+binding+at+higher+temperatures+for+both+polymerases.+At+high+temperatures%2C+DNA+binding+of+Dpo1+monomer+is+slightly+favored+over+binding+of+Dpo4+monomer%2C+but+binding+of+Dpo1+trimer+is+strongly+favored+over+binding+of+Dpo4+dimer%2C+thus+providing+thermodynamic+selection.+%0D%0AThe+results+from+ITC+showed+an+unusually+strong+temperature+dependence+of+the+change+in+heat+capacity+(%E2%88%86C_p%5Eo)%2C+which+switches+from+positive+to+negative+values+with+increasing+temperature.+The+observed+sign+change+in+%E2%88%86C_p%5Eo+does+not+derive+from+temperature-dependent+changes+in+structure%2C+protonation%2C+or+electrostatics.+Rather%2C+we+propose+that+temperature+affects+the+coupled+equilibria+between+self-associations+of+free+Dpo1+or+Dpo4+and+their+binding+to+DNA.+Taken+together%2C+Sso+differentiates+between+Dpo1+and+Dpo4+binding+to+DNA+by+integrating+molecular+and+cellular+principles+including+concentration%2C+temperature%2C+oligomerization%2C+and+coupled+equilibria+to+maintain+uninterrupted%2C+rapid%2C+and+high+fidelity+DNA+replication.&rft.date=2013-10-18&rft.type=University+of+Pittsburgh+ETD&rft.type=PeerReviewed&rft.format=application%2Fpdf&rft.language=en&rft.identifier=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F19672%2F1%2FThesis_HKL_ETD.pdf&rft.identifier=++Lin%2C+Hsiang-Kai++(2013)+Multiequilibria+of+Oligomeric+Thermophilic+DNA+Replication+Polymerases.++Doctoral+Dissertation%2C+University+of+Pittsburgh.++++(Unpublished)++